Msi-specific frameshift peptides (fsp) for prevention and treatment of cancer

ABSTRACT

Described is a vaccine for prevention and treatment of cancer characterized by microsatellite instability (MSI). The vaccine contains an MSI-specific frameshift peptide (FSP) generating humoral and cellular responses against tumor cells or a nucleic acid encoding said FSP. The vaccine of the present invention is particularly useful for the prevention/treatment of colorectal cancer, endometrial cancer, gastric cancer or small bowel cancer.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

All documents cited or referenced herein (“herein cited documents”), andall documents cited or referenced in herein cited documents, togetherwith any manufacturer's instructions, descriptions, productspecifications, and product sheets for any products mentioned herein orin any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. More specifically, all referenced documents areincorporated by reference to the same extent as if each individualdocument was specifically and individually indicated to be incorporatedby reference.

FIELD OF THE INVENTION

The present invention provides a vaccine for prevention and treatment ofcancer characterized by microsatellite instability (MSI). The vaccinemay contain an MSI-specific frameshift peptide (FSP) generating humoraland cellular responses against tumor cells or a nucleic acid encodingsaid FSP.

BACKGROUND OF THE INVENTION

Human tumors develop through two major pathways of genome instability,chromosomal instability and microsatellite instability (MSI) thatresults from defects in the DNA mismatch repair system. MSI isencountered in 15% of colorectal cancers and a variety of extracolonicmalignancies showing a deficient DNA mismatch repair system, includingendometrial cancers, gastric cancers, small bowel cancers and tumors ofother organs. MSI cancers may develop sporadically or in the context ofa hereditary tumor syndrome, hereditary non-polyposis colorectal cancer(HNPCC) or Lynch syndrome.

MSI colorectal cancers are characterized by a high immunogenicity thatresults from the generation of numerous frameshift peptides (FSP) duringthe development of MSI tumors as a direct result of mismatch repairdeficiency leading to alterations of the translational reading framewhen microsatellites in gene-encoding regions are affected by mutation(FIG. 1).

The abundance of predictable MSI-specific FSP antigens and the fact thatthey directly result from the malignant transformation process renderFSP highly promising targets for immune therapy. It is believed that thehuman immune system is a potential resource to eradicate tumor cells andthat effective treatment can be developed if the components of theimmune system are properly stimulated to recognize and eliminate cancercells. Thus, immunotherapy, which may comprise compositions and methodsto activate the body's immune system, either directly or indirectly, toshrink or eradicate cancer, has been studied for many years as anadjunct to conventional cancer therapy.

It is generally admitted that the growth and metastasis of tumorsdepends largely on their capacity to evade host immune surveillance.Most tumors express antigens that can be recognized to a variable extentby the host immune system, but in many cases, the immune response isinadequate. Failure to elicit a strong activation of effector T-cellsmay result from the weak immunogenicity of tumor antigens orinappropriate or absent expression of co-stimulatory molecules by tumorcells. For most T-cells, production of IL-2 and proliferation require aco-stimulatory signal simultaneous with TCR engagement, otherwise,T-cells may enter a functionally unresponsive state, known as clonalanergy.

In spite of the length of time that these therapies have beeninvestigated, there remains a need for improved strategies for enhancingthe immune response against the tumor antigens.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

There is a need in the art for safe and effective compositions that canstimulate the immune system as a cancer immunotherapy.

According to the invention, safe and effective stimulation of the immunesystem as a cancer immunotherapy is achieved by the subject mattersdefined in the claims. In vitro data showed that FSP are highlyimmunogenic and can elicit pronounced FSP-specific T cell responses invitro (Linnebacher et al. 2001, Ripberger et al. 2003, Schwitalle et al.2004). In further studies examining peripheral blood drawn from patientswith MSI colon cancer, a high frequency of FSP-specific T cell responseswas demonstrated (Schwitalle et al. 2008). In spite of the high numberof FSP-specific T cells in the tumor and in the peripheral blood, thesepatients showed no signs of autoimmunity, suggesting that FSPvaccination approaches are not expected to have side effects in terms ofautoimmunity.

Immunological analyses in individuals carrying a germ line mutation inDNA mismatch repair genes predisposing to hereditary non-polyposiscolorectal cancer (HNPCC) were also found to exhibit cellular immuneresponses against FSP, even in the absence of a clinically detectabletumor. This suggests that FSP-specific immune responses may beprotective in HNPCC individuals, suggesting that FSP vaccination mayalso be used in a preventive setting as the first specific preventionapproach in an inherited cancer condition.

In summary:

-   -   (a) Frameshift peptides (FSP) are MSI-specific and directly        result from MSI tumor pathogenesis;    -   (b) No clinically relevant side effects are expected;    -   (c) Combinations of FSPs are predicted to target all tumors with        MSI;    -   (d) FSP vaccination has been designed for therapy of 15% of        colon cancers and tumors of the endometrium, stomach, small        intestine and other organs;    -   (e) Molecular tumor analysis can identify patients that may        benefit from FSP vaccination (targeted therapy); and    -   (f) FSP vaccination may be used as a preventive vaccination in        high risk groups.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIGS. 1A-1B: Schematic illustration of coding microsatellite instabilityresulting from DNA mismatch repair deficiency (Kloor et al., 2010)

Truncated proteins encompassing FSP sequences (red) are generated whencoding microsatellite mutations lead to alterations of the translationalreading frame (example: TGFBR2 protein).

FIG. 2: Exemplary T cell responses against newly designed FSPs inperipheral blood from three patients with MSI colon cancer

FIG. 3: Humoral immune responses against FSPs derived from AIM2(-1),HT001(-1), TAF1B(-1), and TGFBR2(-1)

ELISA revealed FSP-specific antibody responses directed againstneopeptides derived from AIM2(-1), HT001(-1), TAF1B(-1), and TGFBR2(-1).Peptide specificity was demonstrated by preabsorption of respectiveserum antibodies as described previously (Reuschenbach et al., 2008).

FIGS. 4A-4B: Cytotoxic responses as determined by CD107a surfaceexpression

-   -   (A) Significant FSP-specific responses were observed in        different healthy donors after stimulation of T cells with the        antigen for four weeks. Responses did only occur in the presence        of antigen-presenting B cells and the FSP antigen.        Representative responses are shown in bar graphs.    -   (B) representative FACS analysis of CD107a assay for T cells        incubated with B cells as antigen-presenting cells in the        absence (left panel) or presence (right panel) of the FSP        antigen HT001(-1).

DETAILED DESCRIPTION OF THE INVENTION

Thus, the present invention provides a vaccine containing an MSI tumorspecific frameshift peptide (FSP), e.g., derived from TAF1B (Δcc. No.L39061), HT001 (Acc. No. AF113539), AIM2 (Acc. No. AF024714), or TGFBR2(Acc. No. NM_(—)003242) or a nucleic acid encoding said FSP wherein saidFSP is capable of eliciting an immune response against cancer showingMSI.

In a preferred embodiment, the vaccine of the present inventioncontains:

-   -   (a) an FSP which may comprise or consist of the following amino        acid sequence:

NTQIKALNRGLKKKTILKKAGIGMCVKVSSIFFINKQKP; (TAF1B(-1))EIFLPKGRSNSKKKGRRNRIPAVLRTEGEPLHTPSVGMRETTGLGC; (HT001(-1))HSTIKVIKAKKKHREVKRTNSSQLV; (AIM2(-1)) OrASPKCIMKEKKSLVRLSSCVPVALMSAMTTSSSQKNITPAILTCC; (TGFBR2(-1))

-   -   (b) a functional equivalent of an FSP of (b); or    -   (c) a combination of the FSP of (a) and/or (b).

The term “functional equivalent” as used herein relates to, e.g.,variants or fragments of the FSP which are still capable of eliciting animmune response against the cancer, i.e., are still useful as anefficient vaccine. An immune response is defined as a conditionfulfilling at least one of the following criteria: 1. The induction ofCD8-positive T cells, as detectable by cytotoxicity assays or IFN-gammasecretion or perforin expression or granzyme B expression or othercytokines that may be produced by CD8-positive T cells, measurable asabove background by ELISpot or intracellular cytokine staining orcytokine ELISA or equivalent methods. 2. The induction of CD4-positive Tcells, as detectable by cytokine secretion measurable as abovebackground by ELISpot or intracellular cytokine staining or cytokineELISA or equivalent methods. Cytokines may comprise IFN-alpha,IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17,TNF-alpha, TGF-beta or other cytokines that may be produced byCD4-positive T cells. 3. The induction of antibodies, as detectable byWestern blot, ELISA and equivalent or related methods. 4. The inductionof any kind of cellular Immune response not mediated by CD8-positive orCD4-positive T cells as described in 1 and 2.

The variants are characterized by amino acid deletions, substitutions,and/or additions. Preferably, amino acid differences are due to one ormore conservative amino acid substitutions. The term “conservative aminoacid substitutions” involves replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gin, replacement of the basic residuesLys, Arg, and His; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,and Gly.

For the generation of peptides showing a particular degree of identityto the FSP, e.g., genetic engineering may be used to introduce aminoacid changes at specific positions of a cloned DNA sequence to identifyregions critical for peptide function. For example, site directedmutagenesis or alanine-scanning mutagenesis (introduction of singlealanine mutations at every residue in the molecule) may be used(Cunningham and Wells, 1989). The resulting mutant molecules may then betested for immunogenicity using the assay of Example 1.

Preferably, the variants are characterized by not more than 8 aa, morepreferably by not more than 6 as and, even more preferably, by not morethan 4 aa substitutions, deletions and/or additions.

In the fragment of an FSP at least 5 contiguous aa, preferably at least10 contiguous aa, more preferably at least contiguous 15 aa and evenmore preferably at least 20 contiguous aa of the particular amino acidsequence are left. The fragment is still capable of eliciting an immuneresponse.

In a more preferred embodiment, the vaccine of the present inventionadditionally comprises an adjuvant and/or immunostimulatory cytokine orchemokine.

Suitable adjuvants include an aluminium salt such as aluminium hydroxidegel (alum) or aluminium phosphate, but may also be a salt of calcium,iron or zinc, or may be an insoluble suspension of acylated tyrosine, oracylated sugars, cationically or anionicaily derivatisedpolysaccharides, or polyphosphazenes. Other known adjuvants include CpGcontaining oligonucleotides. The oligonucleotides are characterised inthat the CpG dinucleotide is unmethylated. Such oligonucleotides arewell known and are described in, for example WO 96/02555.

The use of immunostimulatory cytokines has become an increasinglypromising approach in cancer immunotherapy. The major goal is theactivation of tumour-specific T lymphocytes capable of rejecting tumourcells from patients with low tumour burden or to protect patients from arecurrence of the disease. Strategies that provide high levels ofimmunostimulatory cytokines locally at the site of antigen havedemonstrated pre-clinical and clinical efficacy. Preferredimmunostimulatory cytokines comprise IL-2, IL-4, IL-7, IL-12, IFNs,GM-CSF and TNF-α.

Chemokines are small (7-16 kD), secreted, and structurally relatedsoluble proteins that are involved in leukocyte and dendritic cellchemotaxis, PMN degranulation, and angiogenesis. Chemokines are producedduring the initial phase of host response to injury, allergens,antigens, or invading microorganisms. Chemokines selectively attractleukocytes to inflammatory foci, inducing both cell migration andactivation. Chemokines may enhance innate or specific host immunityagainst tumors and, thus may also be useful in combination with an FSP.

The vaccine of the present invention might also contain a nucleic acidencoding the FSP for DNA immunization, a technique used to efficientlystimulate humoral and cellular immune responses to protein antigens. Thedirect injection of genetic material into a living host causes a smallamount of its cells to produce the introduced gene products. Thisinappropriate gene expression within the host has importantimmunological consequences, resulting in the specific immune activationof the host against the gene delivered antigen. Direct injection ofnaked plasmid DNA induces strong immune responses to the antigen encodedby the gene vaccine. Once the plasmid DNA construct is injected the hostcells take up the foreign DNA, expressing the viral gene and producingthe FSP inside the cell. This form of antigen presentation andprocessing induces both MHC and class I and class II restricted cellularand humoral immune responses. The DNA vaccines are composed of vectorsnormally containing two unites: the antigen expression unit composed ofpromoter/enhancer sequences, followed by antigen (FSP)-encoding andpolyadenylation sequences and the production unit composed of sequencesnecessary for vector amplification and selection. The construction ofvectors with vaccine inserts is accomplished using recombinant DNAtechnology and the person skilled in the art knows vectors that may beused for this approach. The efficiency of DNA immunization may beimproved by stabilising DNA against degradation, and increasing theefficiency of delivery of DNA into antigen presenting cells. This hasbeen demonstrated by coating biodegradable cationic microparticles (suchas poly(lactide-co-glycolide) formulated with cetyltrimethylammoniumbromide) with DNA. Such DNA-coated microparticles may be as effective atraising CTL as recombinant vaccinia viruses, especially when mixed withalum. Particles 300 nm in diameter appear to be most efficient foruptake by antigen presenting cells.

A variety of expression vectors, e.g., plasmids or viral vectors, may beutilised to contain and express nucleic acid sequences encoding an FSPof the present invention.

A preferred viral vector is a poxvirus, adenovirus, retrovirus,herpesvirus or adeno-associated virus (AAV). Particularly preferredpoxviruses are a vaccinia virus, NYVAC, avipox virus, canarypox virus,ALVAC, ALVAC(2), fowlpox virus or TROVAC.

Recombinant alphavirus-based vectors have also been used to improve DNAvaccination efficiency. The gene encoding the FSP is inserted into thealphavirus replicon, replacing structural genes but leavingnon-structural replicase genes intact. The Sindbis virus and SemlikiForest virus have been used to build recombinant alphavirus replicons.Unlike conventional DNA vaccinations, however, alphavirus vectors areonly transiently expressed. Alphavirus replicons raise an immuneresponse due to the high levels of protein expressed by this vector,replicon-induced cytokine responses, or replicon-induced apoptosisleading to enhanced antigen uptake by dendritic cells.

In a further preferred embodiment, the FSP contains a Tag sequence,preferably at the C-terminus which might be useful for purification of arecombinantly produced FSP. A preferred Tag sequence is a His-Tag. Aparticularly preferred His-Tag consists of 6 His-residues.

The vaccine of the present invention is administered in an amountsuitable for immunization of an individual and, preferably, additionallycontains one or more common auxiliary agents. The employed term “amountsuitable for immunization of an individual” comprises any amount of FSPwith which an individual may be immunized. The amount depends on whetherimmunization is intended as a prophylactic or therapeutic treatment. Inaddition, the individual's age, sex and weight play a role fordetermining the amount. Thus, the amount suitable for immunization of anindividual refers to amounts of the active ingredients that aresufficient to affect the course and the severity of the tumor, leadingto the reduction or remission of such pathology. An “amount suitable forimmunization of an individual” may be determined using methods known toone skilled in the art (see for example, Fingl et al., 1975). The term“individual” as used herein comprises an individual of any kind andbeing able to fall ill with carcinomas. Examples of such individuals arehumans and animals as well as cells thereof.

The administration of the vaccine by injection may be made at varioussites of the individual intramuscularly, subcutaneously, intradermallyor in any other form of application. It may also be favourable to carryout one or more “booster injections” having about equal amounts.

The employed term “common auxiliary agents” comprises any auxiliaryagents suitable for a vaccine to immunize an individual. Such auxiliaryagents are, e.g., buffered common salt solutions, water, emulsions, suchas oil/water emulsions, wetting agents, sterile solutions, etc.

An FSP, nucleic acid sequence or vector of the present invention may bepresent in the vaccine as such or in combination with carriers. It isfavourable for the carriers in the individual not to be immunogenic.Such carriers may be the individual's own proteins or foreign proteinsor fragments thereof. Carriers, such as serum albumin, fibrinogen ortransferrin or a fragment thereof are preferred.

The vaccine of the present invention may be therapeutic, that is, thecompounds are administered to treat an existing cancer, or to preventthe recurrence of a cancer, or prophylactic, that is, the compounds areadministered to prevent or delay the development of cancer. If thecompositions are used therapeutically, they are administered to cancerpatients and are designed to elicit an immune response to stabilize atumor by preventing or slowing the growth of the existing cancer, toprevent the spread of a tumor or of metastases, to reduce the tumorsize, to prevent the recurrence of treated cancer, or to eliminatecancer cells not killed by earlier treatments. A vaccine used as aprophylactic treatment is administered to individuals who do not havecancer, and are designed to elicit an immune response to targetpotential cancer cells.

The present invention also relates to the use of an FSP or functionalequivalent, nucleic acid sequence or vector as defined above for theproduction of a vaccine for the prevention of a carcinoma, e.g.,preventive vaccination of a high risk group, or treatment of acarcinoma. For example, these may be a colorectal cancer, preferably ahereditary non-polyposis colorectal cancer (HNPCC), an endometrialcancer, a gastric cancer or small bowel cancer.

By means of the present invention it is possible to immunizeindividuals, in particular humans and animals. Immunization takes placeby both induction of antibodies and stimulation of CD8+ T cells. Thus,it is possible to take prophylactic and therapeutic steps againstcarcinomas.

The below examples explain the invention in more detail.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1 Detection of FSP-Specific T Cells in Peripheral Bloodfrom Patients with MSI Colon Cancer and Health), HNPCC Mutation Carriers

(A) Methods (ELISpot Assay)

Frequencies of FSP-specific peripheral T cells (pTc) were quantified bydetermining the number of specific IFN-gamma-secreting Tc against newlydesigned FSPs derived from 3 cMS-containing candidate genes usingELISpot analysis. ELISpot assays were performed using 96-wellnitrocellulose plates (Multiscreen; Millipore, Bedford, Mass.) coatedovernight with mouse anti-human IFN-gamma monoclonal antibodies (mAb)(Mabtech, Nacka, Sweden) and blocked with serum containing medium. PTc(day 0, 1×105/well) were plated 6-fold with autologous CD40-activated Bcells (4×10⁴/well, TiBc or pBc, respectively) as antigen-presentingcells in 200 μl IMDM with 10% human AB serum. Peptides were added at afinal concentration of 10 μg/mL. As a positive control, pTc were treatedwith 20 nmoL/L phorbol-12-myristate-13-acetate in combination 350 nmol/Lionomycin. After incubation for 24 hours at 37° C., plates were washedthoroughly, incubated with biotinylated rabbit anti-human IFN-gamma mAbfor 4 hours, washed again, and incubated with streptavidin-alkalinephosphatase for 2 hours, followed by a final wash step. Spots weredetected by incubation with NBT/BCIP (Sigma-Aldrich) for 1 hour,reaction was stopped with water, and, after drying, spots were countedmicroscopically. Methods have been described in detail in Schwitalle etal., 2008.

(B) Results

To examine whether FSP-specific T cell responses were detectable in theperipheral blood of MSI-H CRC patients, ELISpot analyses were performed.Autologous pBc, which showed high expression of MHC class I and II, andcostimulators (CD40, CD80, and CD86) as well as B-cell-specific antigens(CD19 and CD23) were used as antigen-presenting cells.

Pronounced reactivities were observed against newly designed FSPsderived from AIM2(-1), HT001(-1), TAF1B(-1), and TGFBR2(-1):

NTQIKALNRGLKKKTILKKAGIGMCVKVSSIFFINKQKP TAF1B(-1)EIFLPKGRSNSKKKGRRNRIPAVLRTEGEPLHTPSVGMRETTGLGC HT001(-1)HSTIKVIKAKKKHREVKRTNSSQLV AIM2(-1)ASPKCIMKEKKSLVRLSSCVPVALMSAMTTSSSQKNITPAILTCC TGFBR2(-1)

(Neopeptide Sequences are Underlined)

The results obtained from patients (n=8) are summarized in Table 1.Representative ELISpot results are shown in FIG. 2.

TABLE 1 FSP-specific T cell responses against FSPs Patient no TAF1BHT001 AIM2 TGFBR2 ID peptide (−1) (−1) (−1) (−1) MSI01 4 8.17 5.83 3.5 6MSI02 1.5 4.83 7.33 7.17 1.2 MSI03 11.83 28.17 31.83 23.33 10.16 MSI045.5 13 14.17 11.83 8.75 MSI05 3 7.75 16 10 2.8 MSI06 3.17 12 12.33 13.835.2 MSI07 6 17 16.83 14.5 9.33 MC01 1.17 3.5 3.5 3 8 MC02 1.67 2.83 5.834 3.33 MC03 0.83 1.17 1.17 2.17 4.4 MC04 15.17 18.83 18.67 15.17 20.75MC05 0.33 3.17 2.17 1.33 5.67 MC06 30.5 37.83 37.8 34.83 39.5

Mean spot numbers from replicate analyses are given for each peptide andtested individual. MSI01-MSI07—patients with MSI-H CRC,MC01-MC06—healthy HNPCC germ line mutation carrier.

Example 2 Detection of FSP-Specific Humoral Immune Responses inPeripheral Blood from Patients with MSI Colon Cancer and Healthy HNPCCMutation Carriers

(A) Methods (ELISA)

For enzyme-linked immunosorbent assay (ELISA), peptides were coated to96 well polystyrol microtiter plates “Maxisorp” (Nunc, Roskilde,Denmark) at a concentration of 40 μg/ml in PBS overnight at 4° C. Aftercoating, plates were washed 4 times with PBS (0.05% Tween) and blockedfor 1 h with 0.5% casein in PBS. Peptide binding to the microtiterplates and optimal saturating peptide concentration were assessed usingan alkaline phosphatase-peptide competition assay. To monitor individualbackground reactivity of each serum, a control peptide derived from thep16INK4a protein (p16_(—)76-105) was used, against which no antibodyreactivity was found in a large cohort of individuals (Reuschenbach etal., 2008). Each serum was diluted 1:100 in blocking buffer (0.5% caseinin PBS) and tested in duplicates for the presence of antibodies againstall FSPs and the control peptide. As a reference for inter-platevariance, one control serum was included on every plate, and peptidespecific ODs of the control serum were used for normalization. Dilutedsera (50 μl/well) were incubated for 1 h, and after a wash step plateswere incubated with HRP-labeled rabbit anti-human-IgG antibody (JacksonImmunoresearch, West Grove, Pa.; 1:10,000 in blocking buffer) for 1 h.After washing, 50 μl/well of TMB substrate (Sigma, Deisenhofen, Germany)was added and the enzyme reaction was stopped after 30 minutes by adding50 μl/well of 1 N H2SO4. Absorption was measured at 450 nm (referencewavelength 595 nm). Pre-absorption of serum antibodies for specificitycontrol was done according to the method described in detail inReuschenbach et al., 2008.

(B) Results

To examine whether FSP-specific antibodies were detectable in theperipheral blood of MSI-H CRC patients, healthy Lynch syndrome mutationcarriers, and healthy controls ELISA analyses were performed. Pronouncedreactivities were observed against newly designed FSPs derived fromAIM2(-1), HT001(-1), TAF 1B(-1), and TGFBR2(-1). ELISA results are shownin FIG. 3.

Example 3 Detection of FSP-Specific Cytoloxic T Cell Responses

CD107a surface expression on T effector cells upon stimulation with theclinical FSP antigens was measured. CD107a assays are used todemonstrate secretion of cytotoxic granula containing perforin/granzymeB from effector cells. CD107a molecules are expressed on the surface ofcytotoxic granula and become detectable on the cell surface if granulaare released in the context of a cytotoxic T cell response.

To determine the potential of the FSP peptides to induce a cytotoxiccellular immune response, blood was drawn from healthy donors, and Tcells were stimulated with the FSPs using dendritic cells asantigen-presenting cells. Stimulation was repeated weekly and over atime span of four weeks. After four weeks, T cells were harvested andcoincubated with target cells and FSPs CD107a assay was used to analyzepeptide-specific induction of a cytotoxic T cell response.

Cytotoxic responses as determined by CD107a surface expression wereobserved for T cells coincubated with B cells as antigen-presentingcells in the presence of the antigenic FSP (FIG. 4A). Significantresponses were observed in different healthy donors after stimulation ofT cells with the antigen for four weeks. Representative responses areshown in bar graphs. FIG. 4B shows representative FACS analysis ofCD107a assay for T cells incubated with B cells as antigen-presentingcells in the absence (left panel) or presence (right panel) of the FSPantigen HT001(-1).

REFERENCES

-   Cunningham and Wells, Science 244 (1989), 1081-1085.-   Fingl et al., The Pharmocological Basis of Therapeutics, Goodman and    Gilman, eds. Macmillan Publishing Co., New York, pp. 1-46 (1975).-   Kloor M, Michel S, von Knebel Doeberitz M.-   Immune evasion of microsatellite unstable colorectal cancers.-   Int J. Cancer. 2010 Mar. 2. [Epub ahead of print]-   Linnebacher M, Gebert J, Rudy W, Woerner S, Yuan Y P, Bork P, von    Knebel Doeberitz M. Frameshift peptide-derived T-cell epitopes: a    source of novel tumor-specific antigens. Int J. Cancer. 2001 Jul. 1;    93(1):6-11.-   Reuschenbach M, Waterboer T, Wallin K L, Einenkel J, Dillner J,    Hamsikova E, Eschenbach D, Zimmer H, Heilig B, Kopitz J, Pawlita M,    von Knebel Doeberitz M, Wentzensen N.-   Characterization of humoral immune responses against p16, p53, HPV    16 E6 and HPV 16 E7 in patients with HPV-associated cancers.-   Int J. Cancer. 2008 Dec. 1; 123(11):2626-31.-   Reuschenbach M, Kloor M, Morak M, Wentzensen N, Germann A, Garbe Y,    Tariverdian M, Findeisen P, Neumaier M, Holinski-Feder E, von Knebel    Doeberitz M.-   Serum antibodies against frameshift peptides in microsatellite    unstable colorectal cancer patients with Lynch syndrome.-   Fam Cancer. 2009 Dec. 2. [Epub ahead of print]-   Ripberger E, Linnebacher M, Schwitalle Y. Gebert J, von Knebel    Doeberitz M. Identification of an HLA-A0201-restricted CTL epitope    generated by a tumor-specific frameshift mutation in a coding    microsatellite of the OGT gene. J Clin Immunol. 2003 September;    23(5):415-23.-   Schwitalle Y, Kloor M, Eiermann S, Linnebacher M, Kienle P, Knaebel    H P, Tariverdian M, Benner A, von Knebel Doeberitz M. Immune    response against frameshift-induced neopeptides in HNPCC patients    and healthy HNPCC mutation carriers. Gastroenterology. 2008 April;    134(4):988-97.-   Schwitalle Y. Linnebacher M, Ripberger E, Gebert J. von Knebel    Doeberitz M. Immunogenic peptides generated by frameshift mutations    in DNA mismatch repair-deficient cancer cells. Cancer Immun. 2004    Nov. 25; 4:14.

The invention is further described by the following numbered paragraphs:

1. A vaccine containing an MSI-specific frameshift peptide (FSP) whereinsaid FSP is capable of eliciting an immune response against tumorsshowing MSI.

2. The vaccine of paragraph 1 containing

-   -   (a) an FSP comprising the following amino acid sequence:

NTQIKALNRGLKKKTILKKAGIGMCVKVSSIFFINKQKP; (TAF1B(-1))EIFLPKGRSNSKKKGRRNRIPAVLRTEGEPLHTPSVGMRETTGLGC; (HT001(-1))HSTIKVIKAKKKHREVKRTNSSQLV; (AIM2(-1)) orASPKCIMKEKKSLVRLSSCVPVALMSAMTTSSSQKNITPAILTCC; (TGFBR2(-1)) or

-   -   (b) a functional equivalent of an FSP of (b) which is still        capable of eliciting an immune response against cancer showing        MSI; or    -   (c) a combination of FSPs of (a) and/or (b).

3. The vaccine of paragraph 1 or 2, wherein the FSP additionallycontains a Tag sequence.

4. A vaccine containing a nucleic acid sequence encoding the FSP of anyone of paragraphs 1 to 3 or a vector containing said nucleic acidsequence.

5. The vaccine of paragraph 4, wherein said vector is a plasmid or viralvector.

6. The vaccine of paragraph 5, wherein said viral vector is a poxvirus,adenovirus, retrovirus, herpesvirus, alphavirus-based vector oradeno-associated virus (AAV).

7. The vaccine of paragraph 6, wherein said poxvirus is a vacciniavirus, NYVAC, avipox virus, canarypox virus, ALVAC, ALVAC(2), fowlpoxvirus or TROVAC.

8. The vaccine of any one of paragraphs 1 to 7 additionally comprisingan adjuvant and/or immunostimulatory cytokine or chemokine.

9. The vaccine of any one of paragraphs 1 to 8, wherein the activecompound is presented in an oil in water or a water in oil emulsionvehicle.

10. The vaccine of any one of paragraphs 1 to 9 additionally comprisingone or more other antigens.

11. The vaccine according to any one of paragraphs 1 to 10 for use in amethod for the prevention or treatment of a tumor.

12. Use of the MSI tumor specific frameshift peptide (FSP) as defined inany one of paragraphs 1 to 3, the nucleic acid as defined in paragraph4, or the vector as defined in any one of paragraphs 4 to 7 for thepreparation of a vaccine for the prevention or treatment of a tumor.

13. The use according to paragraph 11 or 12 for preventive vaccinationof high risk groups.

14. Use according to any one of paragraphs 11 to 13, wherein the tumoris colorectal cancer, endometrial cancer, gastric cancer or small bowelcancer.

15. Use according to paragraph 14, wherein the colorectal cancer ishereditary non-polyposis colorectal cancer (HNPCC).

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. A vaccine containing an MSI-specific frameshiftpeptide (FSP) wherein said FSP is capable of eliciting an immuneresponse against tumors showing MSI.
 2. The vaccine of claim 1containing (a) an FSP comprising the following amino acid sequence:NTQIKALNRGLKKKTILKKAGIGMCVKVSSIFFINKQKP; (TAF1B(-1))EIFLPKGRSNSKKKGRRNRIPAVLRTEGEPLHTPSVGMRETTGLGC; (HT001(-1))HSTIKVIKAKKKHREVKRTNSSQLV; (AIM2(-1)) orASPKCIMKEKKSLVRLSSCVPVALMSAMTTSSSQKNITPAILTCC; (TGFBR2(-1)) or

(b) a functional equivalent of an FSP of (b) which is still capable ofeliciting an immune response against cancer showing MSI; or (c) acombination of FSPs of (a) and/or (b).
 3. The vaccine of claim 1,wherein the FSP additionally contains a Tag sequence.
 4. A vaccinecontaining a nucleic acid sequence encoding the FSP of claim 1 or avector containing said nucleic acid sequence.
 5. The vaccine of claim 4,wherein said vector is a plasmid or viral vector.
 6. The vaccine ofclaim 5, wherein said viral vector is a poxvirus, adenovirus,retrovirus, herpesvirus, alphavirus-based vector or adeno-associatedvirus (AAV).
 7. The vaccine of claim 6, wherein said poxvirus is avaccinia virus, NYVAC, avipox virus, canarypox virus, ALVAC, ALVAC(2),fowlpox virus or TROVAC.
 8. The vaccine of claim 1 additionallycomprising an adjuvant and/or immunostimulatory cytokine or chemokine.9. The vaccine of claim 1, wherein the active compound is presented inan oil in water or a water in oil emulsion vehicle.
 10. The vaccine ofclaim 1 additionally comprising one or more other antigens.
 11. A methodfor the prevention or treatment of a tumor comprising administering thevaccine of claim 1 to a patient in need thereof.
 12. A method for theprevention or treatment of a tumor comprising preparing a vaccinecomprising administering the vaccine of claim 4 to a patient in needthereof.
 13. The method according to claim 11 wherein the administeringis for preventive vaccination of high risk groups.
 14. The methodaccording to claim 11, wherein the tumor is colorectal cancer,endometrial cancer, gastric cancer or small bowel cancer.
 15. The methodaccording to claim 14, wherein the colorectal cancer is hereditarynon-polyposis colorectal cancer (HNPCC).
 16. The method according toclaim 12 wherein the administering is for preventive vaccination of highrisk groups.
 17. The method according to claim 12 wherein the tumor iscolorectal cancer, endometrial cancer, gastric cancer or small bowelcancer.
 18. The method according to claim 17 wherein the colorectalcancer is hereditary non-polyposis colorectal cancer (HNPCC).